Impact of IL-21 on HIV Vaccine IL-21 is the most recent member of the IL-2 cytokine family that is involved in regulating CD8+ T cell responses, antibody production as well as innate immunity. Using DNA plasmids encoding soluble wild-type and domain-modified Env trimers from the HIV-189.6 isolate and the Gag-pol fusion protein construct consisting of clusters of conserved cytotoxic T lymphocyte (CTL) epitopes, we compared the ability of IL-2-, IL-15-, and IL-21-encoded plasmid DNAs to induce protective immunity against challenge with recombinant vaccinia viruses (rVVs) expressing env, gag and pol gene products in a murine model. Our results demonstrated that IL-21, delivered together with the Env vaccine, had a greater ability than IL-2 and comparable to that of IL-15 to induce protective immunity against challenge with rVV expressing homologous gp!60, though complete eradication of the virus was achieved only in mice treated with both IL-21 and IL-15 at the time of immunization. The protection was associated with elevated levels of Env-specific CTL and NK cell activities in IL-21/IL-15-treated mice, which surpassed those induced by each cytokine alone. The isotype profile of Env-specific antibody responses demonstrated an increased level of IgG1 than IgG2 antibody in mice treated with IL-21 at the time of vaccination, however in mice treated simultaneously with IL-21 and IL-15, IgG1 and IgG2 antibodies were balanced and higher than those elicited by the Env vaccine only. The analysis of kinetics of IL-21-induced protection revealed further increases in the vaccine efficacy when IL-21 plasmid was delivered five days after immunization, consistent with the notion that IL-21 exposure to an immune system that has been primed with an antigen may lead to the optimal augmentation of specific immune responses. We hypothesize that specific combination of IL-21 and IL-15 genes would increase DNA vaccine potency compared with only one of these activities due to the ability of IL-21 to promote IL-15-induced clonal expansion, differentiation, and survival of viral-specific CD8+ T cells, and induce a balanced level of IgG1/IgG2 antibody subclasses. The following specific aims will be pursued: i) To establish the regimen of IL-15 and IL-21 delivery relative to Env and Gag-pol DNA immunizations to maximize the effector differentiation of vaccine-specific immune responses, ii) To determine mechanisms by which IL-21 and IL-15 enhance the induction of memory T cells that provide long-term protection, and iii) To test the effect of optimized cytokine delivery on mucosal immunization and protection against mucosal challenge with rVVs expressing env, gag, or pol gene products. These results may help to optimize AIDS vaccine by exploring novel approaches to maximize the adaptive and innate immune responses in HIV infection.